Mannan-binding lectin (MBL) is an important component of the innate immune system. It is a multimeric protein synthesized in the liver and is one of the C-type lectins, showing calcium-dependent binding to certain carbohydrate (polysaccharide) structures on the surface of microorganisms. Mannan is used as a model of such polysaccharides and MBL shows affinity for monosaccharide components such as N-acetyl-glucosamine and D-mannose. Upon binding to microbial polysaccharides, MBL activates complement by means of associated serine proteases known as MASP-1, MASP-2 and MASP-3, and promotes killing of the microorganism by means of the lytic membrane attack complex and phagocytosis by its opsonizing effect and by direct interaction with putative cell surface receptors.
The MBL molecule is an oligomeric complex of up to six sets of homotrimers of a single chain of 228 amino acid residues. Each chain consists of a 20 amino acid N-terminal cysteine-rich domain followed by a collagen-like domain of 18–20 Gly-Xxx-Yyy repeats, an alpha-helical coiled-coil neck region, and finally, a carbohydrate recognition domain (CRD). Three of these chains form a structural unit or “head”, in which the collagen domains form a triple helix ending in three neck regions that each bears a CRD. The N-terminal domains are linked by interchain disulfide bonds. Up to six such homotrimer heads are then linked via their N-terminal regions to form the structure of the normal MBL molecule, which has been compared to a bunch of three-petalled flowers. This structure is maintained by the formation of disulfide bonds between individual chains of the linked homotrimers. In this form, MBL is capable of associating with the serine proteases MASP-1, MASP-2 and MASP-3 (and with a related non-enzymatic peptide known as MAp19), this complex being capable of activating complement when MBL binds to carbohydrate.
The human MBL gene (mbl2) shows a number of allelic variants. Some occur in the promoter region, the two most significant occurring at positions −550 (H or L) and −221 (Y or X); a further variant occurs in the 5′-untranslated region at position +4 (P or Q); and three occur in exon 1, at position +223 (A or D, Arg52Cys), +230 (A or B, Gly54Asp) and +239 (A or C, Gly57Glu). The promoter haplotypes HY, LY and LX are associated with high, medium and low plasma levels of MBL, respectively, whereas the haplotypes of exon 1 affect the structure and association of the protein chains. Linkage disequilibrium determines that some of the theoretically possible MBL haplotypes are extremely rare and have not been found; among 100 Danish blood donors only the following haplotypes were detected (frequency in brackets): HYPA (0.285), LYQA (0.235), LXPA (0.195), LYPB (0.135), HYPD (0.085), LYPA (0.045), LYQC (0.020). Among A/A genotypes (i.e. with a normal collagenous region), only the LXP/LXP genotypes showed low plasma MBL levels. A/B, A/C and A/D genotypes (i.e. heterozygous for normal and abnormal collagenous regions) showed reduced plasma MBL levels as determined by the old method described; when this was combined with an LX haplotype, even lower levels were recorded. B/B, C/D and D/D genotypes (i.e. with an anomaly of all collagenous regions) showed very low levels of MBL as determined by the old method, even though none of these subjects showed an LX haplotype. The frequencies of the exon 1 haplotypes A, B, C and D in the Danish donors were 0.76, 0.135, 0.020 and 0.085, respectively. This means that the frequencies of A/A, B/B, C/C and D/D genotypes will be the square of these, i.e. affecting 58.76%, 1.82%, 0.04% and 0.72% of the population, respectively (data from Steffensen, R. et al. (2000) J Immunol Methods 241:33–42).
Possible structures of human MBL from B/B, C/C and D/D genotypes have been determined by analyzing the corresponding recombinant proteins expressed in Chinese hamster ovary cells. The results are similar for these three genotypes, and are described with reference to the B/B genotype, responsible for the commonest severe structural abnormality of MBL. Analysis of unreduced MBL from this genotype (known as MBL B) shows that it occurs principally as dimers of two chains, and three such dimers may be linked to form a structure corresponding to two triplet “heads” of normal MBL from A/A genotypes (known as MBL A). Higher oligomers are not formed, and MBL B, whether recombinant or prepared from human donors, associates less strongly with the MASPs, so that it fails to activate complement in in vitro tests.
A/B heterozygotes are believed to possess a mixture of MBL A and B single chains that combine in various ways. Because normal MBL contains up to 18 single chains in six trimeric heads, the proportion of such molecules consisting entirely of normal MBL A chains will be very small in the A/B heterozygotes. The effect of the theoretical 50% admixture of MBL B chains will therefore be to disrupt the structure of the vast majority of the MBL oligomers, so that the B trait is dominant in terms of the MBL phenotype, i.e. the majority of the MBL adopts a structure similar to that of MBL B. The same consideration applies to A/C and A/D heterozygotes, although it appears that the disruptive effect of D chains may be somewhat less than that of B chains.
Existing methods of measuring human MBL depend on the use, in a variety immunoassay designs, of polyclonal and/or monoclonal antibodies raised against MBL. In many cases it is not clear which molecular forms of MBL are preferentially measured by these methods. One commonly used method depends on the use of the mouse monoclonal antibody HYB 131-01 raised against purified human MBL as both capture and detection antibodies in a sandwich ELISA (enzyme-linked immunosorbent assay). In this procedure HYB 131-01 is coated onto microtiter wells so that it can bind MBL in human serum or plasma samples. The amount of MBL bound is a function of the concentration of MBL in the samples. Bound MBL is then quantified by adding labeled HYB 131-01 as a detection antibody. The label can be an enzyme, such as horseradish peroxidase or alkaline phosphatase, capable of producing a quantitative color reaction when incubated with a suitable substrate, or it can be biotin, capable of binding avidin or streptavidin complexed with a suitable enzyme, or it can be europium, to allow detection by time-resolved fluorescence.
These methods also depend on two identical antibody molecules (one to capture and one to detect) binding to a single MBL molecule at two identical but separate sites (epitopes). In practice, this only takes place when the MBL molecule is an oligomer of trimeric subunits, so that only MBL oligomers are measured by these assays. Single or poorly oligomerized MBL trimeric subunits will not permit the simultaneous binding of both capture and detection antibodies. In consequence, these methods cannot adequately measure MBL that is poorly oligomerized, nor can they determine whether the MBL oligomers measured are in fact capable of binding to mannan, one of the defining characteristics of functional MBL. Existing assay methods for determining serum or plasma concentrations of MBL are also incapable of determining whether a low recorded concentration of MBL is due to a low concentration of normal MBL A, as in the LXPA/LXPA genotype, or due to MBL of anomalous structure that does not react adequately in the assay, as in the A/B, A/C, A/D, B/B C/D and D/D genotypes.
However, it is desirable to distinguish between low concentrations in plasma of MBL A and low recorded concentrations that are in fact due to the presence of normal or near-normal concentrations of MBL B, C and D variants. For example, MBL B has been reported to have the same cell-mediated cytotoxic, opsonic and phagocytosis-promoting activities as MBL A. Thus, the failure of the existing assay method to detect MBL B does not reflect these functions of this MBL. In addition, both low plasma concentrations of MBL, as measured by existing immunoassays, and promoter or exon 1 allelic forms associated with such low concentrations, have been correlated with an increased risk of infection in childhood and in immunocompromised patients. They are also correlated with disease progression in chronic granulomatous disease and cystic fibrosis, and with a more severe course of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. As the existing assays have only been able to give a partial picture of a patient's MBL status, it is expected that more precise correlations could be obtained with more differentiated analyses of this status.